Compositions and methods for treating inflammatory related diseases or conditions using Pediococcus acidilactici probiotics

ABSTRACT

The present invention provides a method of treating a disease or condition characterized by inflammation in a subject in need thereof, comprising administering to the subject an effective amount of a  Pediococcus acidilactici  probiotic. Compositions of  Pediococcus acidilactici  probiotic are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/072,308,filed Mar. 16, 2016, in the United States Patent and Trademark Office,and claims the benefit of U.S. Provisional Appl. No. 62/386,347, filedNov. 30, 2015 and U.S. Provisional Appl. No. 62/177,468, filed Mar. 16,2015, which are duly referenced therein according to the requirements of35 U.S.C. § 119(e)(1). The content of the aforesaid provisionalapplications are relied upon and incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The field of the invention relates to compositions and uses ofPediococcus acidilactici bacteria.

BACKGROUND OF THE INVENTION

Probiotics are beneficial microorganisms naturally existing ingastrointestinal (GI) tracts of humans and animals. In 2001, the WorldHealth Organization defined probiotics as “Live microorganisms, which,when administered in adequate amounts, confer a health benefit on thehost” (Joint FAO/WHO Expert Consultation Report, 2001). Manyprobiotics-related health benefits such as decreasing symptoms fromantibiotics-induced diarrhea, acute diarrhea, traveler diarrhea,allergy, respiratory and urinary tract infections, inflammatory boweldisease, irritable bowel syndrome, colon and bladder cancer, andrheumatoid arthritis have been reported. Conventionally, Bifidobacteriumand Lactobacillus are commercially available probiotics. However, thesebacteria are sensitive to air exposure, elevated temperature, andstomach acids.

Pediococcus acidilactici is a plant based probiotic that is widelyapplied in sausage preparation for human consumption and as animal feedadditives to improve animal health. Moreover, P. acidilactici wasreported to be able to stimulate the antibody production againstparasitic infection of broiler chicken coccidiosis, and ovalbuminantibody production in ovalbumin vaccinated horses (Furr et al., Journalof Equine Veterinary Science, 34:1156-1163 (2014)). Both T-cell andB-cell multiplication were detected in rats fed with Pediococcus-basedprobiotics which were mixtures of P. acidilactici and Saccharomycesboulardii.

This background information is provided for informational purposes only.No admission is necessarily intended, nor should it be construed, thatany of the preceding information constitutes prior art against thepresent invention.

SUMMARY OF THE INVENTION

It is to be understood that both the foregoing general description ofthe embodiments and the following detailed description are exemplary,and thus do not restrict the scope of the embodiments.

According to non-limiting example embodiments, in one aspect, theinvention provides a method of treating a disease or conditioncharacterized by inflammation in a subject in need thereof, comprisingadministering to the subject an effective amount of a Pediococcusacidilactici probiotic.

In some embodiments, the administration of Pediococcus acidilacticiprobiotic increases the number of anti-inflammatory M2 macrophage cellsin the subject, thereby treating the disease or condition. In someembodiments, the administration of Pediococcus acidilactici probioticincreases IL-10 production in the subject, thereby treating the diseaseor condition. In some embodiments, the administration of Pediococcusacidilactici probiotic decreases the levels of IL-6 and/or IL-23 in thesubject, thereby treating the disease or condition.

In some embodiments, the disease or condition characterized byinflammation is selected from the group consisting of malignancy(cancer), arthritis, cardiovascular disease, hepatitis, infection, woundhealing, pancreatitis, gastroesophageal reflux disease, diabetes,inflammatory bowel disease, peptic ulcer disease, bronchitis,cholecystitis, appendicitis, bursitis, dermatitis, asthma, autoimmunedisease, pelvic inflammatory disease, gout, trauma, foreign bodyinfection, burns, dental work, tendonitis, rhinitis, mucositis, andexposure to toxins such as chemicals and alcohol.

In some embodiments, the Pediococcus acidilactici probiotic is strainNRRL B-50517. In some embodiments, the subject is a human.

In some embodiments, the subject is administered greater than 1.0×10⁹cfu of the probiotic. In some embodiments, the subject is administeredgreater than 4.0×10⁹ cfu of the probiotic.

In some embodiments, the subject is administered one or more additionaltherapeutic agents. In some embodiments, the subject is administered oneor more chemotherapeutic (anti-cancer) agents and/or radiotherapy incombination with the Pediococcus acidilactici probiotic.

In some embodiments, the subject is not administered another therapeuticagent. In some embodiments, the subject is not administered anotherprobiotic.

In another aspect, the invention provides a composition comprising aPediococcus acidilactici probiotic. In some embodiments, the Pediococcusacidilactici is strain NRRL B-50517. In some embodiments, thecomposition is a pharmaceutical composition.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary, and thus do notrestrict the scope of the invention. Other objects, features andadvantages of the present invention will become apparent from thefollowing detailed description. It should be understood, however, thatthe detailed description and the specific examples, while indicatingspecific embodiments of the invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 . Effect of P. acidilactici 50517 probiotic on body fat %.

FIG. 2 . Effect of P. acidilactici 50517 probiotic on IL-23 activity.

FIG. 3 . Effect of P. acidilactici 50517 probiotic on IL-6 activity.

FIG. 4 . Effects of Pediococcus-based probiotics on a dog withpancreatitis. A 14 years old, female, spayed toy poodle withpancreatitis was treated 100 mg KAMOSTAAL100 twice a day at a point intime (red dot), and the treatment was stopped about 2½ months later (redsquare). Pediococcus-based probiotics were applied (green dot), andstopped at about a month later (green square). After the relapse,Pediococcus-based probiotics were applied again (green circle), andcontinued for a period of time (green arrow).

FIG. 5 . Pediococcus acidilactici NRRL B-50517 manufacture processsummary flow chart.

DETAILED DESCRIPTION

The invention is based, in part, on the surprising discovery thatadministration of effective amounts of a Pediococcus acidilacticiprobiotic are able to treat diseases or conditions characterized byinflammation.

Reference will now be made in detail to embodiments of the inventionwhich, together with the drawings and the following examples, serve toexplain the principles of the invention. These embodiments describe insufficient detail to enable those skilled in the art to practice theinvention, and it is understood that other embodiments may be utilized,and that structural, biological, and chemical changes may be madewithout departing from the spirit and scope of the present invention.Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art.

For the purpose of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example in thedocument where the term is originally used). The use of the word “a” or“an” when used in conjunction with the term “comprising” in the claimsand/or the specification may mean “one,” but it is also consistent withthe meaning of “one or more,” “at least one,” and “one or more thanone.” The use of the term “or” in the claims is used to mean “and/or”unless explicitly indicated to refer to alternatives only or thealternatives are mutually exclusive, although the disclosure supports adefinition that refers to only alternatives and “and/or.” As used inthis specification and claim(s), the words “comprising” (and any form ofcomprising, such as “comprise” and “comprises”), “having” (and any formof having, such as “have” and “has”), “including” (and any form ofincluding, such as “includes” and “include”) or “containing” (and anyform of containing, such as “contains” and “contain”) are inclusive oropen-ended and do not exclude additional, unrecited elements or methodsteps. Furthermore, where the description of one or more embodimentsuses the term “comprising,” those skilled in the art would understandthat, in some specific instances, the embodiment or embodiments can bealternatively described using the language “consisting essentially of”and/or “consisting of” As used herein, the term “about” means at mostplus or minus 10% of the numerical value of the number with which it isbeing used.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Current Protocols in Molecular Biology(Ausubel et. al., eds. John Wiley & Sons, N.Y. and supplements thereto),Current Protocols in Immunology (Coligan et al., eds., John Wiley StSons, N.Y. and supplements thereto), Current Protocols in Pharmacology(Enna et al., eds. John Wiley & Sons, N.Y. and supplements thereto) andRemington: The Science and Practice of Pharmacy (Lippincott Williams &Wilicins, 2Vt edition (2005)), for example.

I. Methods

According to non-limiting example embodiments, in one aspect, theinvention provides a method of treating a disease or conditioncharacterized by inflammation in a subject in need thereof, comprisingadministering to the subject an effective amount of a Pediococcusacidilactici probiotic.

As used herein, “treat” and all its forms and tenses (including, forexample, treating, treated, and treatment) refers to therapeutictreatment. In certain aspects of the invention, those in need oftreatment include those already with a pathological disease or conditionof the invention (including, for example, a cancer), in which casetreating refers to administering to a subject (including, for example, ahuman or other mammal in need of treatment) a therapeutically effectiveamount of a composition so that the subject has an improvement in a signor symptom of a pathological condition of the invention. The improvementmay be any observable or measurable improvement. Thus, one of skill inthe art realizes that a treatment may improve the patient's condition,but may not be a complete cure of the disease or pathological condition.

The subject to be treated herein is not limiting. In some embodiments,the subject to be treated is a mammal, bird, reptile or fish. Mammalsthat can be treated in accordance with the invention, include, but arenot limited to, humans, dogs, cats, horses, mice, rats, guinea pigs,sheep, cows, pigs, monkeys, apes and the like, subject to a disease orother pathological condition characterized by inflammation. The term“patient” and “subject” are used interchangeably. In some embodiments,the subject is a human.

The Pediococcus acidilactici probiotic can be administered 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 times per day. In some embodiments, thePediococcus acidilactici probiotic is administered 4 times a day, 2times a day, or once per day. In some embodiments, the Pediococcusacidilactici probiotic is administered every 2 hours, every 4 hours,every six hours, every 8 hours, every 10 hours, every 12 hours or every24 hours. In some embodiments, the Pediococcus acidilactici probiotic isadministered once a day.

The duration of administration of the Pediococcus acidilactici probioticcan vary for each individual to be treated/administered depending on theindividual cases and the diseases or conditions to be treated. In someembodiments, the Pediococcus acidilactici probiotic can be administeredcontinuously for a period of several days, weeks, months, or years oftreatment or can be intermittently administered where the individual isadministered the Pediococcus acidilactici probiotic for a period oftime, followed by a period of time where they are not treated, and thena period of time where treatment resumes as needed to treat the diseaseor condition. For example, in some embodiments, the individual to betreated is administered the Pediococcus acidilactici probiotic of theinvention daily, every other day, every three days, every four days, 2days per week 3 days per week, 4 days per week, 5 days per week or 7days per week. In some embodiments, the individual is administered thePediococcus acidilactici probiotic for 1 week, 2 weeks, 3 weeks, 4weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 1 year or longer.

In some embodiments, the administration of Pediococcus acidilacticiprobiotic increases the number of anti-inflammatory M2 macrophage cellsin the subject, thereby treating the disease or condition. In someembodiments, the anti-inflammatory M2 macrophage cells increase by about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 100%, about 125%, about 150%, about 175%,about 200%, about 250%, about 300%, about 350%, about 400%, or about450%, about 500%, about 600%, about 700%, about 800%, about 900% orabout 1000% or more over untreated levels.

In some embodiments, the administration of Pediococcus acidilacticiprobiotic increases IL-10 production in the subject, thereby treatingthe disease or condition. In some embodiments, the IL-10 productionincreases by about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 100%, about 125%,about 150%, about 175%, about 200%, about 250%, about 300%, about 350%,about 400%, or about 450%, about 500%, about 600%, about 700%, about800%, about 900% or about 1000% or more over untreated levels.

In some embodiments, the administration of Pediococcus acidilacticiprobiotic decreases the levels of IL-6 and/or IL-23 in the subject,thereby treating the disease or condition. In some embodiments, thelevels of IL-6 and/or IL-23 decrease by about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 100% over untreated levels.

In some embodiments, the administration of Pediococcus acidilacticiprobiotic increases the number of anti-inflammatory M2 macrophage cellsin the subject, in combination with increasing IL-10 production, andoptionally decreasing IL-6 and/or IL-23 in the subject.

In some embodiments, the disease or condition characterized byinflammation is selected from the group consisting of malignancy(cancer), arthritis, cardiovascular disease, hepatitis, infection, woundhealing, pancreatitis, gastroesophageal reflux disease, diabetes,inflammatory bowel disease, peptic ulcer disease, bronchitis,cholecystitis, appendicitis, bursitis, dermatitis, asthma, autoimmunedisease, pelvic inflammatory disease, gout, trauma, foreign bodyinfection, burns, dental work, tendonitis, rhinitis, mucositis, andexposure to toxins such as chemicals and alcohol.

As used herein, “cancer” refers to a pathophysiological conditionwhereby cells are characterized by dysregulated and/or proliferativecellular growth and the ability to induce said growth, which includesbut is not limited to, carcinomas and sarcomas, such as, for example,acute lymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcancer, AIDS-related cancers, AIDS-related lymphoma, anal cancer,astrocytoma (including, for example, cerebellar and cerebral), basalcell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumor (including, for example, ependymoma,meduUoblastoma, supratentorial primitive neuroectodermal, visual pathwayand hypothalamic glioma), cerebral astrocytoma/malignant glioma, breastcancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoidtumor (including, for example, gastrointestinal), carcinoma of unknownprimary site, central nervous system lymphoma, cervical cancer, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-Cell lymphoma, endometrial cancer, ependymoma, esophageal cancer,Ewing's Family of tumors, extrahepatic bile duct cancer, eye cancer(including, for example, intraocular melanoma, retinoblastoma,gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (GIST), germ cell tumor (including, forexample, extracranial, extragonadal, ovarian), gestational trophoblastictumor, glioma, hairy cell leukemia, head and neck cancer, squamous cellhead and neck cancer, hepatocellular cancer, Hodgkin's lymphoma,hypopharyngeal cancer, islet cell carcinoma (including, for example,endocrine pancreas), Kaposi's sarcoma, laryngeal cancer, leukemia, lipand oral cavity cancer, liver cancer, lung cancer (including, forexample, non-small cell), lymphoma, macroglobulinemia, malignant fibroushistiocytoma of bone/osteosarcoma, meduUoblastoma, melanoma, Merkel cellcarcinoma, mesothelioma, metastatic squamous neck cancer with occultprimary, mouth cancer, multiple endocrine neoplasia syndrome, multiplemyeloma/plasma cell neoplasm, mycosis fungoides, myelodysplasiasyndromes, myelodysplastic/myeloproliferative diseases, myeloma, nasalcavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,non-Hodgkin's lymphoma, oral cancer, oral cavity cancer, osteosarcoma,oropharyngeal cancer, ovarian cancer (including, for example, ovarianepithelial cancer, germ cell tumor), ovarian low malignant potentialtumor, pancreatic cancer, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pineoblastoma and supratentorial primitive neuroectodermal tumors,pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonaryblastoma, pregnancy and breast cancer, primary central nervous systemlymphoma, prostate cancer, rectal cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterinesarcoma, Sezary syndrome, skin cancer (including, for example,non-melanoma or melanoma), small intestine cancer, supratentorialprimitive neuroectodermal tumors, T-Cell lymphoma, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter, trophoblastictumor (including, for example, gestational), unusual cancers ofchildhood and adulthood, urethral cancer, endometrial uterine cancer,uterine sarcoma, vaginal cancer, viral induced cancers (including, forexample, HPV induced cancer), vulvar cancer, Waldenstrom'smacroglobulinemia, Wilms' Tumor, and women's cancers.

The Pediococcus acidilactici probiotic that can be used in accordancewith the invention is not limiting. In some embodiments, the Pediococcusacidilactici is a strain that is viable above 65° C., is able to grow inaerobic and anaerobic conditions, and in a pH range between 1 and 6.2.In some embodiments, the Pediococcus acidilactici is a strain depositedin the Agricultural Research Service (ARS) Patent Culture Collection asNRRL B-50517. The address of the ARS depository is 1815 N. UniversityStreet, Peoria, IL 61604. The deposited material has been accordedaccession number NRRL B-50517 and was deposited on May 24, 2011. StrainNRRL B-50517 is described in U.S. application Ser. No. 13/676,579, whichis herein incorporated by reference.

In some embodiments, the Pediococcus acidilactici for use in theinvention can be selected for tolerance to elevated temperatures, low pHvalues, and aerobic and anaerobic conditions.

In accordance with the invention, a “therapeutically effective amount”or “effective amount” is administered to the subject. As used herein a“therapeutically effective amount” or “effective amount” is an amountsufficient to decrease, suppress, or ameliorate one or more symptomsassociated with the disease or condition. In some embodiments, thesubject is administered greater than 1.0×10⁹ cfu of the probiotic. Insome embodiments, the subject is administered greater than 4.0×10⁹ cfuof the probiotic.

In some embodiments, the subject is administered one or more additionaltherapeutic agents. In some embodiments, the one or more additionaltherapeutic agents are those commonly used to treat the disease orcondition characterized by inflammation.

In some embodiments, the subject is administered in combination ananti-inflammatory drug. In some embodiments, the administeredPediococcus acidilactici and anti-inflammatory drug act synergistically.In some embodiments, the anti-inflammatory drug is a non-steroidalanti-inflammatory drug (NSAID). In some embodiments, anti-inflammatorydrug is selected from the group consisting of Antazoline, Balsalazide,Beclometasone, Betamethasone, Budesonide, Celecoxib, Colchicine,Deflazacort, Dexamethasone, Dexibuprofen, Diclofenac, Etanercept,Etodolac, Felbinac, Fenoprofen, Flumetasone, Fluorometholone,Flurbiprofen, Flurbiprofen, Fluticasone, Gentamicin, Hydrocortisone,Ibuprofen, Indometacin, Ketoprofen, Loteprednol, Mefenamic acid,Meloxicam, Mesalazine, Methylprednisolone, Mometasone, Nabumetone,Naproxen, Nepafenac, Olsalazine, Prednisolone, Rimexolone,Sulfasalazine, Sulindac, Tenoxicam, Tiaprofenic acid, Triamcinolone andcombinations thereof.

In some embodiments, the subject is administered one or more anti-canceragents and/or radiotherapy in combination with the Pediococcusacidilactici probiotic to treat cancer in the subject.

In some embodiments, the anti-cancer agent is selected from the groupconsisting of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane(Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE,ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-TrastuzumabEmtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil(Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara(Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium),Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil),Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant,Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin(Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra(Ofatumumab), Asparaginase Erwinia chrysanthemi, Avastin (Bevacizumab),Axitinib, Azacitidine, BEACOPP, Becenum (Carmustine), Beleodaq(Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Bevacizumab,Bexarotene, Bexxar (Tositumomab and I 131 Iodine Tositumomab),Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto(Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, BrentuximabVedotin, Busulfan, Busulfex (Busulfan), Cabazitaxel,Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar (IrinotecanHydrochloride), Capecitabine, CAPDX, Carboplatin, CARBOPLATIN-TAXOL,Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant,Casodex (Bicalutamide), CeeNU (Lomustine) Ceritinib, Cerubidine(Daunorubicin Hydrochloride), Cervarix (Recombinant HPV BivalentVaccine), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP,Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex(Clofarabine), Clolar (Clofarabine), CMF, Cometriq(Cabozantinib-S-Malate), COPP, COPP-ABV, Cosmegen (Dactinomycin),Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza(Ramucirumab), Cytarabine, Cytarabine, Liposomal, Cytosar-U(Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine,Dacogen (Decitabine), Dactinomycin, Dasatinib, DaunorubicinHydrochloride, Decitabine, Degarelix, Denileukin Diftitox, Denosumab,DepoCyt (Liposomal Cytarabine), DepoFoam (Liposomal Cytarabine),Dexrazoxane Hydrochloride, Docetaxel, Doxil (Doxorubicin HydrochlorideLiposome), Doxorubicin Hydrochloride, Doxorubicin HydrochlorideLiposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome(Dacarbazine), Efudex (Fluorouracil), Elitek (Rasburicase), Ellence(Epirubicin Hydrochloride), Eloxatin (Oxaliplatin), Eltrombopag Olamine,Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride, EPOCH,Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), ErlotinibHydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Etopophos(Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet(Doxorubicin Hydrochloride Liposome), Everolimus, Evista (RaloxifeneHydrochloride), Exemestane, Fareston (Toremifene), Faslodex(Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (FludarabinePhosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil),Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI,FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn(Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPVQuadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine),Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride,GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin,Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate),Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer(Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (EribulinMesylate), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant,HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine,Recombinant, Hycamtin (Topotecan Hydrochloride), Hyper-CVAD, Ibrance(Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (PonatinibHydrochloride), Idamycin (Idarubicin Hydrochloride), IdarubicinHydrochloride, Idelalisib, Ifex (Ifosfamide), Ifosfamide, Ifosfamidum(Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod,Inlyta (Axitinib), Intron A (Recombinant Interferon Alfa-2b), Iodine 131Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), IrinotecanHydrochloride, Istodax (Romidepsin), Ixabepilone, Ixempra (Ixabepilone),Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla(Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride),Kepivance (Palifermin), Keytruda (Pembrolizumab), Kyprolis(Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lenalidomide,Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole,Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Levulan(Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (DoxorubicinHydrochloride Liposome), Liposomal Cytarabine, Lomustine, Lupron(Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), LupronDepot-Ped (Leuprolide Acetate), Lupron Depot-3 Month (LeuprolideAcetate), Lupron Depot-4 Month (Leuprolide Acetate), Lynparza(Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane(Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megace(Megestrol Acetate), Megestrol Acetate, Mekinist (Trametinib),Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide),Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate),Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride,Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen(Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran(Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin),Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized NanoparticleFormulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar(Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate),Nilotinib, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate(Romiplostim), Obinutuzumab, OEPA, Ofatumumab, OFF, Olaparib,Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ontak (DenileukinDiftitox), Opdivo (Nivolumab), OPPA, Oxaliplatin, Paclitaxel, PaclitaxelAlbumin-stabilized Nanoparticle Formulation, PAD, Palbociclib,Palifermin, Palonosetron Hydrochloride, Pamidronate Di sodium,Panitumumab, Paraplat (Carboplatin), Paraplatin (Carboplatin), PazopanibHydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron(Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Di sodium, Perj eta(Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin),Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), PonatinibHydrochloride, Pralatrexate, Prednisone, Procarbazine Hydrochloride,Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (EltrombopagOlamine), Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan(Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride,Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant HumanPapillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus(HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV)Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib,R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan(Rituximab), Rituximab, Romidepsin, Romiplostim, Rubidomycin(Daunorubicin Hydrochloride), Ruxolitinib Phosphate, SclerosolIntrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot(Lanreotide Acetate), Sorafenib Tosylate, Sprycel (Dasatinib), STANFORDV, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib),Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (PeginterferonAlfa-2b), Sylvant (Siltuximab), Synovir (Thalidomide), Synribo(Omacetaxine Mepesuccinate), TAC, Tafinlar (Dabrafenib), Talc, TamoxifenCitrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride),Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel),Taxotere (Docetaxel), Temodar (Temozolomide), Temozolomide,Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thiotepa, Toposar(Etoposide), Topotecan Hydrochloride, Toremifene, Torisel(Temsirolimus), Tositumomab and I 131 Iodine Tositumomab, Totect(Dexrazoxane Hydrochloride), TPF, Trametinib, Trastuzumab, Treanda(Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide), Tykerb(Lapatinib Ditosylate), Vandetanib, VAMP, Vectibix (Panitumumab), VeIP,Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (VinblastineSulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate),Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (VincristineSulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, VinorelbineTartrate, VIP, Vismodegib, Voraxaze (Glucarpidase), Vorinostat, Votrient(Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori(Crizotinib), Xeloda (Capecitabine), XELIRI, Xgeva (Denosumab), Xofigo(Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab),Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin (IbritumomabTiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept,Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat),Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), andZytiga (Abiraterone Acetate). In some embodiments, the drug is selectedfrom the group consisting of Paclitaxel, Curcumin, Docetaxel,Ixabepilone, Vinblastine, Colchicine, Y-27632 Fasudil, SU6656 Dasatinib,HDAC inhibitors, ROCK inhibitors, Parthenolide, Costunolide and ML-7Jazplakinolide.

In some embodiments, the subject is not administered another therapeuticagent and is administered a composition consisting of or consistingessentially of the Pediococcus acidilactici probiotic.

In some embodiments, the subject is administered one or more additionalprobiotics. In some embodiments, the subject is not administered anotherprobiotic.

II. Compositions

In some embodiments, the invention provides a composition comprising aPediococcus acidilactici probiotic. In some embodiments, the compositioncomprises Pediococcus acidilactici NRRL B-50517. In some embodiments,the compositions comprise effective amounts of Pediococcus acidilactici,including Pediococcus acidilactici NRRL B-50517.

In some embodiments the compositions are pharmaceutical compositions. Insome embodiments, the compositions are pharmaceutical compositionscomprising effective amounts of Pediococcus acidilactici, includingPediococcus acidilactici NRRL B-50517 which are capable of treating ofone or more diseases or conditions characterized by inflammation.

In some embodiments, the pharmaceutical composition comprises one ormore pharmaceutically acceptable carriers or excipients.Pharmaceutically acceptable carriers and excipients are those that arecompatible with the other ingredients in the formulation andbiologically acceptable. The Pediococcus acidilactici can be provided incombination with a pharmaceutically acceptable carrier, excipients ordiluent. Suitable carriers, excipients and/or diluents include, but arenot limited to, pharmaceutical grade starch, mannitol, lactose,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol,detergents, emulsifiers or water (preferably sterile). The compositionmay be a mixed preparation of a composition or may be a combinedpreparation for simultaneous, separate or sequential use (includingadministration). The Pediococcus acidilactici can also be administeredin sachets that have to be added to a glass of water and then drunk.

In some embodiments, the composition is suitable for oraladministration. In some embodiments, the composition is a tablet,capsule, pill, dragee, suspension, lozenge, emulsion, aqueous solution,liquid, gel, or syrup. In some embodiments, the compositions can bedelivered in the form of functional foods and/or beverages, as well asin the form of various supplements.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the Pediococcus acidilactici;as a powder or granules, which in some embodiments can be wettable,spray-dried or freeze-dried; as a solution or a suspension in an aqueousor non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion.

In some embodiments, a tablet can be made by compression or molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g. povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g. sodium starch glycollate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

In some embodiments, the composition comprises one or more of thefollowing: tablets, pills, capsules, ovules, solutions or suspensions,which may contain flavouring or colouring agents, for immediate-,delayed-, modified-, sustained-, pulsed- or controlled-releaseapplications.

For aqueous suspensions and/or elixirs, the composition of the presentinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, propylene glycol and glycerin, andcombinations thereof.

In some embodiments, the compositions of the invention are formulated inunit dosage form for ease of administration and uniformity of dosage.The term “unit dosage form” as used herein refers to physically discreteunits suited as unitary dosages for the individuals to be treated. Insome embodiments, the compositions are formulated into discrete dosageunits each containing a predetermined “unit dosage” or “unit dose” ofone or more active compounds calculated to produce the desired effect inassociation with the required pharmaceutical carrier.

In some embodiments, the composition comprises gelatin capsules. In someembodiments, the gelatin capsules comprise effective amounts of P.acidilactici NRRL B-50517 fermentative cultures with peach fruit powder,in a dose of from about 1-4 billion CFU.

While is it possible to administer Pediococcus acidilactici aloneaccording to the present invention, the Pediococcus acidilactici aretypically administered on or in a support as part of a product, inparticular as a component of a food product, a dietary supplement or apharmaceutical formulation. These products typically contain additionalcomponents well known to those skilled in the art.

In one embodiment, the Pediococcus acidilactici are employed accordingto the invention in a food product such as a food supplement, a drink ora powder based on milk. Here, the term “food” is used in a broadsense—and covers food for humans as well as food for animals (i.e. afeed). In one embodiment, the food is for human consumption.

The food may be in the form of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

When used as, or in the preparation of, a food, such as functional food,the composition of the present invention may be used in conjunction withone or more of: a nutritionally acceptable carrier, a nutritionallyacceptable diluent, a nutritionally acceptable excipient, anutritionally acceptable adjuvant, a nutritionally active ingredient.

By way of example, the composition of the present invention can be usedas an ingredient to soft drinks, a fruit juice or a beverage comprisingwhey protein, health teas, cocoa drinks, milk drinks, yoghurt anddrinking yoghurt, cheese, ice cream, water ices and desserts,confectionery, biscuits cakes and cake mixes, snack foods, balancedfoods and drinks, fruit fillings, care glaze, chocolate bakery filling,cheese cake flavoured filling, fruit flavoured cake filling, cake anddoughnut icing, instant bakery filling creams, fillings for cookies,ready-to-use bakery filling, reduced calorie filling, adult nutritionalbeverage, acidified soy/juice beverage, aseptic/retorted chocolatedrink, bar mixes, beverage powders, calcium fortified soy/plain andchocolate milk, calcium fortified coffee beverage.

The composition can further be used as an ingredient in food productssuch as American cheese sauce, anti-caking agent for grated & shreddedcheese, chip dip, cream cheese, dry blended whip topping fat free sourcream, freeze/thaw dairy whipping cream, freeze/thaw stable whippedtopping, low fat and light natural cheddar cheese, low fat Swiss styleyoghurt, aerated frozen desserts, hard pack ice cream, label friendly,improved economics & indulgence of hard pack ice cream, low fat icecream: soft serve, barbecue sauce, cheese dip sauce, cottage cheesedressing, dry mix Alfredo sauce, mix cheese sauce, dry mix tomato sauceand others.

The term “dairy product” as used herein is meant to include a mediumcomprising milk of animal and/or vegetable origin. As milk of animalorigin there can be mentioned cow's, sheep's, goat's or buffalo's milk.As milk of vegetable origin there can be mentioned any fermentablesubstance of vegetable origin which can be used according to theinvention, in particular originating from soybeans, rice or cereals.

In some embodiments, the food product employed according to theinvention is a fermented milk or humanized milk.

In some embodiments, the compositions can be used in connection withyoghurt production, such as fermented yoghurt drink, yoghurt, drinkingyoghurt, cheese, fermented cream, milk based desserts and others.

Suitably, the composition can be further used as an ingredient in one ormore of cheese applications, meat applications, or applicationscomprising protective cultures.

The present invention also provides a method of preparing a food or afood ingredient, the method comprising admixing the compositionaccording to the present invention with another food ingredient.

In some embodiments, the present invention relates to products that havebeen contacted with the composition of the present invention (andoptionally with other components/ingredients), wherein the compositionis used in an amount to be capable of improving the nutrition and/orhealth benefits of the product.

As used herein the term “contacted” refers to the indirect or directapplication of the composition of the present invention to the product.Examples of the application methods which may be used, include, but arenot limited to, treating the product in a material comprising thecomposition, direct application by mixing the composition with theproduct, spraying the composition onto the product surface or dippingthe product into a preparation of the composition.

Where the product of the invention is a foodstuff, the composition ofthe present invention is preferably admixed with the product.Alternatively, the composition may be included in the emulsion or rawingredients of a foodstuff. In a further alternative, the compositionmay be applied as a seasoning, glaze, colorant mixture, and the like.

For some applications, it is important that the composition is madeavailable on or to the surface of a product to be affected/treated. Thisallows the composition to impart one or more of the following favourablecharacteristics: nutrition and/or health benefits.

The compositions of the present invention may be applied to intersperse,coat and/or impregnate a product with a controlled amount of a viablemicroorganism.

In some embodiments, the composition is used to ferment milk or sucrosefortified milk or lactic media with sucrose and/or maltose where theresulting media containing all components of the composition—i.e. saidmicroorganism according to the present invention—can be added as aningredient to yoghurt milk in suitable concentrations—such as forexample in concentrations in the final product which offer a daily doseof 10⁶-10¹⁰ cfu. The microorganism according to the present inventionmay be used before or after fermentation of the yoghurt.

In some aspects, the microorganisms according to the present inventionare used as, or in the preparation of, animal feeds, such as livestockfeeds, in particular poultry (such as chicken) feed, pet food, or pettreats.

In some embodiments, where the product is a food product, thePediococcus acidilactici should remain effective through the normal“sell-by” or “expiration” date during which the food product is offeredfor sale by the retailer. Preferably, the effective time should extendpast such dates until the end of the normal freshness period when foodspoilage becomes apparent. The desired lengths of time and normal shelflife will vary from foodstuff to foodstuff and those of ordinary skillin the art will recognize that shelf-life times will vary upon the typeof foodstuff, the size of the foodstuff, storage temperatures,processing conditions, packaging material and packaging equipment.

In some embodiments, the composition of the present invention may beused as a food ingredient and/or feed ingredient. As used herein theterm “food ingredient” or “feed ingredient” includes a formulation whichis or can be added to functional foods or foodstuffs as a nutritionalsupplement. The food ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

In some embodiments, the composition of the present invention may be—ormay be added to—food supplements (also referred to herein as dietarysupplements).

In some embodiments, the composition of the present invention may be—ormay be added to—functional foods. As used herein, the term “functionalfood” means food which is capable of providing not only a nutritionaleffect, but is also capable of delivering a further beneficial effect toconsumer. Accordingly, functional foods are ordinary foods that havecomponents or ingredients (such as those described herein) incorporatedinto them that impart to the food a specific functional—e.g. medical orphysiological benefit—other than a purely nutritional effect. Althoughthere is no legal definition of a functional food, most of the partieswith an interest in this area agree that they are foods marketed ashaving specific health effects beyond basic nutritional effects. Somefunctional foods are nutraceuticals. Here, the term “nutraceutical”means a food which is capable of providing not only a nutritional effectand/or a taste satisfaction, but is also capable of delivering atherapeutic (or other beneficial) effect to the consumer. Nutraceuticalscross the traditional dividing lines between foods and medicine.

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not to be construed as a limitation thereof.

EXAMPLES Example 1—P. acidilactici Administration Stimulates InnateImmune Responses in Animals

This example describes the effect of administering P. acidilactici oninnate immune responses in rats.

TABLE 1 Stimulation of macrophage activities on rats fed withPediococcus-based probiotics MAC* Control 4.0 ± 0.3 Low 4.0 ± 0.5 Medium6.0 ± 1.6 High 7.2 ± 1.5 *Sprague Dawley rats (9 weeks) were fed Harlan#7012 rat chow ad libitum. The probiotic was Pediococcus-basedprobiotics (Imagilin, Frederick, MD). There were four groups (n = 10):control group (no probiotic); low dose (1 × 10⁹ cfu); mid group dose (2× 10⁹); and high dose (10 × 10⁹). Animals were given 2 grams of foodmixed with probiotic at 11 a.m. Then from 8 pm to 11 am, chow suppliedad libitum. Water was ad libitum 24 h. Tail blood samples were analyzedfor complete blood counts.

When the rats were administered 2.0×10⁹ to 10×10⁹ cfu Pediococcus-basedprobiotics per day for 15 days, the amounts of macrophages increased150% to 180% comparing to those from rats without probiotics. Theincrease of macrophages indicates that Pediococcus-based probiotics canstimulate rat innate immune responses. Interestingly, when rats were fedwith low amounts (1.0×10⁹) of Pediococcus-based probiotics, the numberof macrophages were similar to the amounts of macrophages as those fromthe control. This indicates that adequate amounts of Pediococcus-basedprobiotics are required to stimulate innate immune responses, such asincreasing the amounts of macrophage cells.

Example 2—P. acidilactici Administration Stimulates Cytokine Productionin Human Subjects

This example describes the effect of administering P. acidilactici oncytokine production in human subjects.

For innate immune responses, macrophages are broadly divisible into twogroups: pro-inflammatory M1 macrophages and anti-inflammatory M2macrophages. The M2 macrophages also refers to macrophages that functionin constructive processes like wound healing and tissue repair, andthose that turn off damaging immune system activation by producinganti-inflammatory cytokines like interleukin-10 (IL-10).

TABLE 2 Increase of interleukin-10 (IL-10) on Pediococcus probioticstreated human volunteer subjects. Detection of IL-6 Detection of IL-10 %of IL-6 After treated % of IL-10 Before treated After treated aftertreated Before treated Pediococcus after treated Participant PediococcusPediococcus Pediococcus Pediococcus probiotics for Pediococcus IDprobiotics probiotics probiotics probiotics 45 days probiotics AB01 0.500.29 58% 1.99 3.99 200% AB02 1.77 0.93 53% 4.44 8.16 183% AB03 1.94 1.7289% 5.66 8.95 158% AB04 0.72 0.93 129%  4.44 8.79 198% AB05 0.12 0.19158%  0.77 3.25 422% Average 97.4% Average 232% * Serum samples werecollected from five volunteers before administration of Pediococcusacidilactici NRRL B-50517 probiotics, and after administration of 4billion cfu of Pediococcus probiotics per day for 45 days. Serum sampleswere analyzed using Luminex-based multiplex assays (EMD Millipore;Milliplex) designed to measure biomarkers associated withpro-inflammatory IL-6 and anti-inflammatory IL-10.

All five volunteers exhibited significantly increased anti-inflammatoryIL-10 activity (from 158% to a 422% increase) after administration ofPediococcus probiotics for 45 days. On the contrary, the effect onpro-inflammatory IL-6 showed inconsistent results, which exhibiteddecreased activity in three volunteers and increased activity in twovolunteers. These results demonstrate that administration of Pediococcusprobiotics in human subjects may enhance more than two fold theanti-inflammatory IL-10 activity. These results, together with theresults showing increases of macrophages in Pediococcus-based probioticstreated rats indicate that Pediococcus-based probiotics can enhanceinnate immunity of humans and animals. The innate immune responses ofhumans and animals treated with Pediococcus exhibit increases of M2macrophage and anti-inflammatory IL-10.

Example 3—Effects of P. acidilactici NRRL B-50517 Supplementation forUse in Weight Management: A Controlled, Randomized, Double-Blind Trial

This weight management study assessed the effect of a 12 weeksupplementation of Pediococcus acidilactici NRRL B-50517 probioticstrain on 30 adult participants in a controlled, randomized, doubleblind trial. Percent body fat was measured at the beginning and end ofthe trial with bioelectric impedance analysis (BIA). Levels ofproinflammatory biomarkers interleukin-6 (IL-6) and interleukin-23(IL-23) were determined using blood samples collected before the trialbegan and after it concluded. Appetite, energy level, bowel movement,stool quality, bloating, and gas, were monitored throughout the studyusing weekly questionnaires. The specific weight loss andanti-inflammatory effect of P. acidilactici is described here for thefirst time. Daily supplementation with 4 billion CFU P. acidilacticiresulted in on average, the probiotic group lost 0.86±0.42% percent bodyfat whereas the control group gained 0.28%±0.19, p=0.0264.Pro-inflammatory IL-6 ratios differed by 0.61±0.22 and 3.06±0.87 inprobiotic and control groups, respectively (p=0.0295); pro-inflammatoryIL-23 ratio was 0.65±0.14 in the probiotic and 1.71±0.38 in the controlgroups, p=0.0068.

Methods and Materials

Participants in the study were selected on a volunteer basis;distribution of age, sex, and BMI was equal across treatment groups.Volunteers were not instructed to alter their regular dietary patternsor exercise routines during the study. The probiotic was tested in agroup of subjects divided as such: 20% normal weight status(18.5-24.99), 47% overweight (25-29.99), and 33% obese (>30).

Prior to the beginning of the supplementation period, participantsunderwent an extensive physical exam including a bioelectric impedanceanalysis to determine body fat percentage and blood work to quantifyIL-6 and IL-23 levels. The same exam procedure was repeated at theconclusion of the study. Over the course of 12 weeks, 30 participantswere administered either 2 gelatin capsules containing a compound of P.acidilactici NRRL B-50517 fermentative cultures with peach fruit powdersonce daily, amounting to a dose of 4 billion CFU Pediococcusprobiotics/day, or a placebo treatment of 2 capsules containing onlypeach powder.

The safety of the probiotic was analyzed in terms of impact on appetite,energy level, bowel movement, stool quality, bloating, and gas. As partof a weekly questionnaire, participants were asked to score theirexperience of these symptoms on an arbitrary scale from 1 to 5, 1 beingthe least severe and 5 being the most severe.Results

The present results show for the first time that supplementation of theprobiotic Pediococcus acidilactici NRRL B-50517 to the diet ofoverweight and obese individuals alters bodily fat storage andinfluences concentration of inflammatory biomarkers linked to thepathology of obesity. The clear difference between % body fat, IL-6 andIL-23 levels observed between placebo and 50517-treated groups at theconclusion of the study demonstrate the effect of the probiotic tosupport weight loss even without traditional dietary modification orexercise. Results are shown in FIGS. 1-3 . Error bars for all threegraphs reflect that the respective values of percent body fat, IL-6, andIL-23 for the placebo and probiotic treated groups are not within onestandard deviation of one another and thus signal a significantdifference between the two.

Shown in FIG. 1 is the effect of P. acidilactici 50517 probiotic on bodyfat %. Participants were administered either 2 gelatin capsulescontaining a compound of P. acidilactici NRRL B-50517 fermentativecultures with peach fruit powders once daily, amounting to a dose of 4billion CFU Pediococcus probiotics/day, or 2 capsules containing onlypeach powder. % body fat was determined by bioelectric impedanceanalysis (BIA) test. Values are based on double blind field evaluationsof Pediococcus probiotics on total 30 volunteers for 3 months treatment.P=0.0264, t=2.4073, placebo mean=0.280.+−0.0.190 (SEM); NutriLots.TM.mean=−0.864.+−0.0.418 (SEM). The significantly higher difference inpercent body fat observed in the probiotic-treated group (NutriLots) ascompared to the placebo group after 3 months of treatment supports thatP. acidilactici NRRL B-50517 supplementation can result in acceleratedweight loss with no changes to diet or exercise patterns.

Impact of Pediococcus-Probiotic Supplementation on InflammatoryBiomarkers IL-6 and IL-23.

Shown in FIG. 2 is the effect of P. acidilactici 50517 probiotic onIL-23. Blood samples were collected from each participant either withplacebo or with probiotics before the study began and after thetreatment period concluded to determine changes in IL-6 and IL-23presence. Marked decreases in both IL-6 and IL-23 were observed in thePediococcus-probiotic treated group. Values are based on double blindfield evaluations of Pediococcus probiotics on total 30 volunteers for 3months treatment. P=0.0295, t=2.4239, placebo mean=3.058.+−0.0.867(SEM); NutriLots.TM. mean=−0.612.+−.0.221 (SEM). The lower ratio ofIL-23 in the probiotic-treated group (NutriLots) suggests that 50517 iscapable of reducing obesity-related inflammation.

Shown in FIG. 3 is the effect of P. acidilactici 50517 Probiotic onIL-6. Values are based on double blind field evaluations of Pediococcusprobiotics on total 30 volunteers for 3 months treatment. P=0.0068,t=3.0194, placebo mean=1.714.+−.0.377 (SEM); NutriLots.TM.mean=−0.648.+−.0.137 (SEM). The decreased ratio of IL-6 in the probiotictreated group (NutriLots) indicates that 50517 is capable of reducingobesity-related inflammation.

Discussion

In the present study, the 12-week P. acidilactici NRRL B-50517 probiotictreatment produced significant decreases in body fat percent,interleukins 6 and 23 when administered to participants of varying BMI(FIGS. 1,2, and 3 ). Consistent results across the board indicate thatthe means of P. acidilactici action is not limited exclusively toindividuals of obese weight status, but for those who are of lower BMIas well. A majority of previous studies have demonstrated the efficacyof LAB probiotic treatment on solely obese subjects. Where otherprobiotic strains were ineffective in reducing in the presence ofobesity-related inflammation, 50517 decreased levels of bothinterleukins 6 and 23 as compared to the placebo group.

Safety of the probiotic was confirmed in a separately published studyconducted alongside the present research. No significant difference inparticipant scores of appetite, bowel movement, bloating, stool quality,energy level, or gas was observed between the beginning and conclusionof the trial period in either the placebo or probiotic-treated group.

These findings have tremendous implications for future treatment andprevention of metabolic disease. As a large percentage of the cases ofchronic conditions such as cardiovascular disease (CVD) and type 2diabetes are developed in tandem with obesity, improving the managementof this one disease has the potential to considerably reduce theincidence of several other prominent threats to public health.

While previously thought to only act as storage vessels for excesscalories in the form of triglycerides, adipocytes have been discoveredto play a complex role in metabolism, immunity, and cancer (Calabro P,Yeh ETH. 2007. Obesity, Inflammation, and Vascular Disease: the role ofthe adipose tissue as an endocrine organ. Subcellular Biochemistry.42:63-91). White adipose cells secrete proteins including cytokines andhormone-like factors such as adiponectin, leptin, and resistin; thisphenomenon is of particular interest because of the involvement of thesemolecules in vascular and metabolic complications (Calabro P, Yeh ETH.2007. Obesity, Inflammation, and Vascular Disease: the role of theadipose tissue as an endocrine organ. Subcellular Biochemistry.42:63-91). In a majority of obese patients, low grade inflammation ofwhite adipose tissue (WAT) resulting from chronic activation of innateimmunity poses an increased possibility of insulin resistance, impairedglucose tolerance, and eventual development of diabetic tendencies(Bastard J P, Maachi M, Lagathu C, Kim M J, Caron M, Vidal H, Capeau J,Feve B. 2006. Recent advances in the relationship between obesity,inflammation, and insulin resistance. Eur. Cyt. Net. 17(1): 4-12).Macrophage infiltration of obese WAT acts as a source ofpro-inflammatory cytokines, further contributing to the pathogenesis ofinsulin resistance. Meanwhile, circulating levels of adiponectin, aninsulin-sensing effector highly expressed in WAT, are lower in obesethan normal weight subjects. The WAT in these individuals overproducesand secretes increased levels of numerous inflammatory moleculesincluding IL-6, another modulator of insulin sensitivity. Thus, thepro-inflammatory pathogenesis of obesity and systemic development ofinsulin resistance are closely entwined, linked by the modulation ofWAT.

Regulation of calorie extraction from dietary substances could beconsidered a possible mechanism of probiotic action for the resultsshown here. The composition of human gut microbiota has beenconsistently implicated as a determinant of body weight as a result ofits critical role in nutrient acquisition and energy harvest andregulation (Tennyson C A, Friedman G. 2008. Microecology, obesity, andprobiotics. Curr. Opin. Endocr. Diab. Obes. 15(5):422-7; DiBaise J K,Zhang H, Crowell M D, Krajmalnik-Brown R, Decker G A, Rittmann B E.2008. Gut microbiota and its possible relationship with obesity. MayoClinic Proceedings. 83(4):460-69). There is reason to believe thattargeted microbial community moderation through the introduction of aprobiotic could then have potential as a novel therapeutic agent in thetreatment of metabolic disease. Conscious editing of the microbiome maybe the key to reconcile the imbalance between energy intake andexpenditure attributed to the obese state.

Unchanged reported scores for appetite in both the placebo andprobiotic-treated groups indicate no potential alterations in dietarypatterns or influence on satiety hormone leptin.

In decreasing body fat percent while simultaneously lowering serum IL-6concentration, it can be presumed that P. acidilactici 50517 mayincrease insulin sensitivity and decrease overall systemic inflammation,therefore contributing to lowered risk of type 2 diabetes. As IL-6 isalso linked to vascular damage in obese individuals, a lowered serumlevel of the cytokine would be likely to reduce risk of CVD (Calabro P,Yeh ETH. 2007. Obesity, Inflammation, and Vascular Disease: the role ofthe adipose tissue as an endocrine organ. Subcellular Biochemistry.42:63-91). Inflammatory activity in obese individuals, increasing inaccordance with WAT macrophage infiltration, can be assumed to decreaseupon a loss in body fat.

The results shown here mirror those observed in an analysis ofpost-surgical results in morbidly obese individuals. Following bariatricsurgery, patients have shown clinically relevant decreases in IL-6,triglycerides, cholesterol, LDL, glucose, and insulin correlated to BMI,validating the existence of a relationship between weight and theinflammatory profile, and further elucidating that between BMI andbiochemical parameters of chronic metabolic and vascular conditions(Illan-Gomez F, Gonzalvez-Ortega M, Orea-Soler I, Alcaraz-Tafalla M S,Aragon-Alonso A, Pascual-Diaz M, Perez-Paredes M, Lozano-Almela M L.2012. Obesity and inflammation: change in C-reactive protein, tumornecrosis factor-alpha and interleukin-6 after bariatric surgery. Obes.Surg. 22:950-55).

The marked decrease in IL-23 concentration observed in theprobiotic-treated group is also a powerful signifier of lowered diseaserisk. IL-23/IL-17 is strongly associated with activation of signalpathways leading to tumor formation and the pathway for carcinogenesis.Because stimulation of the IL-23/IL-17 axis has been observed in obesewomen independent of increases in abdominal fat, insulin resistance,leptin, or MIF levels, it is reasonable to assume that dietary andbehavioral patterns associated with the development of obesity, and notthe obese state itself, may be responsible (Sumarac-Dumanovic M,Stevanovic D, Ljubic A, Jorga J, Simic M, Stamenkovic-Pejkovic D,Starcevic V, Trajkovic V, Micic D. 2009. Increased activity ofinterleukin-23/interleukin-17 proinflammatory axis in obese women. Int.J. Obes. 33:151-56).

Conclusions

In summary, the probiotic P. acidilactici NRRL B-50517 showed loweringeffects on body fat percent, IL-6, and IL-23, suggesting its beneficialinfluence on weight management and metabolic disease. In light of theevidence set forth in this study, Pediococcus acidilactici NRRL B-50517could prove to be effective in reduction of body fat and inflammationamong those individuals seeking to lose weight.

Example 4—Effects of Pediococcus Based Probiotics on Dogs and Cats withPancreatitis

A 14 years old, female, spayed toy poodle with significant abdominalpain, vomiting and drop in appetite was diagnosed to possibly sufferfrom pancreatitis. The images of the ultrasound were shown to have highfats echo from the stomach to the duodenum, and the results of the serumanalysis were shown to have spec cPL (Specific Canine Pancreatic Lipase)of 432 ug/L. Although the dog was switched to a low fat diet and treated100 mg KAMOSTAAL100 twice a day immediately, the spec cPL remained highabove 400 and reach 610 ug/L about 2.5 months thereafter with diarrheaand vomiting. At that point, the dog was treated twice a day of 200 mgPediococcus-based probiotics together with current treatment of low fatdiet and twice a day 100 mg KAMOSTAAL100. Interestingly, not onlydiarrhea and vomiting were stopped, the spec cPL was back to normal at163 ug/L within about 6 weeks. Therefore the treatment of bothPediococcus-based probiotics and KAMOSTAAL100 was stopped. However,about three months later, the dog had a relapse with the spec cPLincreased to 276 ug/L. At this time, the dog was treated wasPediococcus-based probiotics 200 mg twice a day only. The treatment wascontinued for about 7 months with good control of diarrhea, vomiting,and loss of appetite, and the spec cPL was shown to be normal at 108ug/L (FIG. 4 ).

The spec cPL (Specific Canine Pancreatic Lipase) and the spec fPL(Specific Feline Pancreatic Lipase) are the normal spec cPL of canineand are the well-established assays for pancreatitis in dogs and cats.In healthy dogs and cats, the spec cPL is <200 ug/L, and the spec fPL is0.7-3.5 ug/L. Dogs and cats are regarded to have pancreatitis, when speccPL is greater than 400 ug/L and the cat spec fPL is >5.4 ug/L. Based onthis criteria, we applied Pediococcus-based probiotics on two dogs andone cat, of which both dogs had spec cPL >600 ug/L, and the cat had specfPL 50 ug/L, suffering from pancreatitis. All of these dogs and cats,not only the vomiting and diarrhea were stopped but also the spec cPLand spec fPL were controlled and returned to normal. Moreover, two dogswith possible pancreatitis were also treated, since they had elevatedspec cPL (303 ug/L and 205 ug/L).

Example 5—Effects of Pediococcus Based Probiotics on the Dogs withCancers Under Chemotherapy Treatment

Four dogs having various cancers and that were undergoing chemotherapywere treated with doses of Pediococcus probiotics. After a short periodof treatment with the Pediococcus probiotics, the dogs experiencedimproved symptoms.

Side Body effects weight Drugs for before Dose of Days of Age (kg) SexCancer chemotherapy treatment Pediococcus treatment Improvement 16 6Spayed Breast panriifu (anti- Diarrhea, 2 billion 3 days After cancer-breast cancer lost cfu/day administered surgery 3rd drug: Tamoxifen)appetite Pediococcus time based probiotics with anti- cancer drug for 3days, vomiting stopped, lost appetite recovered. 5 16.2 Castratedlymphoma Adriamycin General 4 billion 7 days Good clinical cfu/daycondition is good 12 15.7 Castrated anal Carboplatin Soft stool 4billion 3 days Good cystoma cfu/day 10 35 female lymphoma L-asparaginasevomiting, 8 billion 4 days Good after 4 (Leunase), diarrhea cfu/day daysof Chlorambucil, treatment, Prednisolone, diarrhea Ulcerlmin recovered(Sucralfate hydrate) * The chemotherapy treatment and Pediococcus basedprobiotics administration treatment were performed at Daktari AnimalHospital Central, Torri, and Yaizu.

Example 6—Manufacture of Pediococcus acidilactici NRRL B-50517

First the strain needed for the fermentation process is selected fromthe bacteria being cryoed at −70° C. freezer. Grow the culture insterile media bottles. When grown, pull samples to verify cleanlinessand basic phenotypical purity. If determined to be clean and the cellsmatch initial gram stain smears, approval to make the media(fermentation broth) for the tanks is given. Prior to inoculation, theinoculum tank is CIP'd (Clean In Place) with caustic and acid solutions.We sanitize the tanks prior to filling them with the broth fermentationingredients which are dumped, mixed, and sterilized. The tank media issterilized at 220° F.-250° F. for thirty minutes to an hour and a halfdepending on the volume of the tank. We then bring down the temperatureto 85-95° F. to seed the tank with the inoculum. When the inoculationtank is grown, we cool the tank to 55-65° F. When cool, a sample istaken for repeated purity checks. If approved for release then, werepeat the steps for tank preparation and inoculation. Production willinoculate the tanks with the grown and approved inoculum bottles. Afterall the desired tanks are grown, we repeat the purity checks prior toprepping the centrifuge and concentrating the cells into a condensedliquid. The condensed liquid culture is put into a sterile holding tank.We add sterile liquid cryoprotectant solution to the centrifugedculture.

It is homogenized in the holding tank by the agitator. When homogenous,the culture is pumped into a sterile kettle (which is a functionalaliquot for the cryofreezing or pelletizing of the product) to bepelletized in a liquid nitrogen vat. When complete the frozen pelletsare lyophilized or freeze dried. After drying, we mill the freeze driedpellets into a fine powder. We will take the ground culture andhomogenize it to ensure uniformity prior to sampling the culture for thequality assurance tests by morphological, physiological, 16S rRNA DNAsequences, and high temperature stress assays. Product is removed fromthe blender that was used to homogenize the material and then bag andstore it at cool area, room temperatures.

Example 7—Formulation and Testing of Pediococcus Acidilactici NRRLB-50517 in Food

As knowledge of the health benefits of probiotics spreads and the demandfor probiotic-infused food products continues to rise, food corporationsare faced with a new set of challenges as they begin to collaborate withbiotechnology companies. First, they must select one or more probioticstrains from the plethora of available options. Ideally, the chosenbacteria would need to: 1. Survive any manufacturing stress such as highheat treatment, 2. Possess compatibility with the chemical and physicalproperties of the desired food matrix, 3. Maintain viability in the foodfor the duration of the product's shelf life once incorporated, and 4.Resist destruction by digestive mechanisms in order to confer its healthbenefits to the host.

Many probiotic strains popular in commercial supplements (such asLactobacillus and Bifidobacterium) do not effectively fulfill theserequirements and are thus unsuitable for industrial food production.Lacking the critical high heat resistance necessary to survive inrecently pasteurized food, the applications of the two lactic acidbacteria (LAB) are severely limited in this context. The instability ofthese strains at room temperature would present additional complicationsin transport and storage for both food retailers and potentialconsumers. As facultative anaerobes to obligate anaerobes, Lactobacillusand Bifidobacterium would be especially vulnerable to losses inviability upon any exposure to oxygen, further reducing their potentialfor incorporation to food products. A more versatile, reliable strain isrequired to formulate effective probiotic-infused food.

Pediococcus acidilactici NRRL B-50517 is a uniquely formulated powdercomposed of the strain of bacteria capable of withstanding greatvariation in temperature, osmotic pressure, and oxygen exposure. Adurable microorganism originally isolated from plant material, theprobiotic has proven ability to survive in a wide range of food productsunder varying environmental conditions and heat treatment procedures.

Survival of P. acidilactici NRRL B-50517 in sucrose solutions ranging inconcentration from 10 to 50% is indicative of probiotic resistance toosmotic pressure (Table 3). Where weaker bacteria would likely loseviability in a solution with comparably high osmolality, P. acidilacticiNRRL B-50517 retains remarkably steady cell counts even at the highesttested concentration. Comparable results were obtained in solutions oflactose within the same concentration range over the course of 9 days(Table 4). In solutions of sterile water, 0.1 to 20% NaCl, and combinedsolutions of NaCl and sucrose, P. acidilactici NRRL B-50517 maintainedsignificant cell viability in all assayed samples for up to one week,showcasing probiotic ability to adapt to a myriad of chemicalenvironments (Table 5).

TABLE 3 Survival of P. acidilactici NRRL B-50517 incubated in highconcentrated sucrose solution stored at room temperature Numbers of P.acidilactici NRRL B-50517 viable cells after different incubation timein sucrose solution at room temperature % Sucrose solution Control 1 day3 days 10% 3.00E+08 5.20E+09 N/A 20% 3.50E+08 3.00E+08 1.40E+08 30%3.30E+08 2.10E+08 1.50E+08 40% 5.10E+08 3.70E+08 3.10E+08 50% 6.50E+084.70E+08 3.30E+08 *: 0.2 g 1Billion (1B) CFU/g P. acidilactici NRRLB-50517 was added to 20 mL of each sucrose solution and stored at roomtemperature. Viability tests were conducted by serially dilutingsucrose + P. acidilactici NRRL B-50517 solution in 0.1% saline, platingonto MRS, and enumerating plates after overnight incubation. **plateswere contaminated on the third day, preventing continued testing forstability. Conclusions: P. acidilactici NRRL B-50517 maintains viabilityin sucrose solutions ranging in concentration from 10-50%, indicatingresistance of P. acidilactici NRRL B-50517 to high osmotic pressureenvironment

TABLE 4 Survival of P. acidilactici NRRL B-50517 incubated in highconcentrated lactose solution stored at room temperature Numbers of P.acidilactici NRRL B-50517 viable cells after different incubation timein lactose solution at room temperature % Lactose Control 1 day 3 days 7days 9 days 10% 1.20E+08 2.10E+08 1.90E+08 2.00E+08 8.00E+07 20%1.80E+08 1.40E+08 2.40E+08 2.10E+08 2.00E+08 30% 2.30E+08 1.60E+084.30E+08 2.00E+08 1.60E+08 40% 4.90E+08 3.70E+08 3.30E+08 3.67E+094.00E+08 50% 1.80E+08 7.00E+07 1.00E+08 1.00E+09 1.50E+08 *: 0.2 g 1 B/gP. acidilactici NRRL B-50517 was added to 20 mL of each lactose solutionand stored at room temperature. Viability tests were conducted byserially diluting sucrose + P. acidilactici NRRL B-50517 solution in0.1% saline, plating onto MRS, and enumerating plates after overnightincubation. Conclusions: Over a period of 9 days, P. acidilactici NRRLB-50517 retained highly stable viable cell counts in 10% to 50% lactosesolutions.

TABLE 5 Survival of P. acidilactici NRRL B-50517 incubated in high saltsolution stored at room temperature Numbers of P. acidilactici NRRLB-50517 viable cells after different incubation time in salt solution atroom temperature Solution 5 min 30 min 120 min 7 days 14 days 22 days 35days Sterile H2O 4.13E+08 4.31E+08 4.03E+08 1.01E+08 4.90E+05 0.00E+000.00E+00 only 0.1% NaCl 3.88E+08 3.82E+08 3.72E+08 1.82E+08 9.80E+071.60E+07 1.10E+05 1% NaCl 4.38E+08 3.98E+08 3.51E+08 1.82E+08 2.90E+079.00E+04 5.00E+03 2.5% NaCl 4.98E+08 3.87E+08 4.02E+08 2.21E+08 5.70E+071.00E+04 0.00E+00 5% NaCl 4.85E+08 3.40E+08 3.87E+08 1.39E+08 1.25E+082.90E+07 1.01E+06 7.5% NaCl 3.82E+08 3.72E+08 4.60E+08 6.70E+07 4.20E+072.90E+07 7.12E+06 10% NaCl 4.32E+08 4.08E+08 4.96E+08 4.70E+07 7.00E+063.00E+06 2.60E+05 20% NaCl 4.53E+08 3.62E+08 5.36E+08 1.03E+08 5.10E+073.20E+07 4.52E+06 25% Sucrose, 3.92E+08 3.68E+08 4.80E+08 9.00E+075.60E+07 9.00E+06 1.56E+06 10% NaCl * 0.2 g 1 B/g P. acidilactici NRRLB-50517 was added to 20 mL of different concentrations of NaCl orsucrose + NaCl solution and stored at room temperature. Viability testswere conducted by serially diluting NaCl + P. acidilactici NRRL B-50517solution in 0.1% saline, plating onto MRS, and enumerating plates afterovernight incubation.

[in relevant part]

When assayed extensively for viability, 50517 retained viable cellcounts (CFU/g) for up to 113 days after incorporation to peanut butterwhen stored at room temperature, indicating high shelf stability (Table6). The probiotic showed similarly successful results when heated to 85°C. in peanut butter with storage at room temperature thereafter.Stability was similarly constant when the probiotic was incorporated to85° C.-heated pudding; cell counts remained within one log over a periodof 29 days with refrigerator storage (Table 7).

TABLE 6 Integration of Pediococcus acidilactici NRRL B-50517 intocommercial peanut butter Numbers of viable cells after integrated P.acidilactici NRRL B-50517 into peanut butter stored at differenttemperature Days of Room storage temperature % Survival 37° C. %Survival Control 3.15E+10 100.00 3.15E+10 100.00 0 2.78E+10 88.18 n/an/a 7 1.34E+10 42.68 1.63E+10 51.85 14 2.33E+10 74.07 1.78E+10 56.44 222.91E+10 92.24 1.49E+10 47.27 113 4.78E+09 15.17 2.00E+07 0.06 *:Samples of P. acidilactici NRRL B-50517 and peanut butter were preparedby mixing 6 g of 100 B/g P. acidilactici NRRL B-50517 powder with 20 gpeanut butter, then stored at either room temperature (23° C.) or 37° C.Stability tests were conducted by adding 0.1 g of the mixture to 5 mL0.1% saline, serially diluting the solution for plating onto MRS, andenumerating plates after overnight incubation at 45° C. Percent survivalwas calculated as a fraction of a P. acidilactici NRRL B-50517 + salinecontrol (0.2 g 100 B/g P. acidilactici NRRL B-50517 added to 10 mL 0.1%saline at room temperature). Conclusion: P. acidilactici NRRL B-50517shows high cell counts (CFU/g) in peanut butter at room temperature overa period of 113 days, indicating that a product containing bothingredients would maintain high shelf stability. Even when stored at 37°C., the peanut butter and P. acidilactici NRRL B-50517 mixture displayssimilarly high viability over 22 days, dropping off between the 22 and113 day viability tests.

TABLE 7 Survival of P. acidilactici NRRL B-50517 in peanut butter afterhigh temperature (85° C.) treatment Numbers of viable cells after heattreated integrated P. acidilactici NRRL B-50517 into peanut butter at85° C. and stored at room temperature Peanut Butter Day 1 % Survival Day7 % Survival Day 14 % Survival Control 3.15E+10 100.00 3.15E+10 100.003.15E+10 100.00 PB 1 7.98E+09 25.33 1.01E+09 3.22 2.22E+09 7.04 PB 21.38E+10 43.83 3.17E+09 10.05 3.67E+09 11.66 Hazelnut 2.53E+08 0.807.66E+08 2.43 8.11E+08 2.57 * Samples of P. acidilactici NRRL B-50517and peanut butter were prepared by mixing 1.2 g of 100 B/g P.acidilactici NRRL B-50517 powder into 3.8 g peanut butter. Empty tubeswere heated to 85° C. before 0.5 g peanut butter and P. acidilacticiNRRL B-50517 mixture was added and left on the hot plate for 5 min.After a 10 min cooling period, 10 mL 0.1% saline was added to each tube.Viability tests were conducted by serially diluting into saline, platingonto MRS, and enumerating plates after overnight incubation at 45° C.Percent survival was calculated as a fraction of a P. acidilactici NRRLB-50517 + saline control (0.2 g 100 B/g P. acidilactici NRRL B-50517added to 10 mL 0.1% saline at room temperature). Conclusion: Over aperiod of two weeks after high heat treatment, P. acidilactici NRRLB-50517 maintained high viability in nut butters, supportingcompatibility of the P. acidilactici NRRL B-50517 with commerciallyproduced nut products. PB1 ingredients: roasted peanuts, sugar,hydrogenated vegetable oil (cottonseed, soybean, and rapeseed oil) toprevent separation, salt. Hazelnut Spread ingredients: sugar, vegetableoil (palm and rapeseed oil), hazelnuts, cocoa powder, skim milk, whey,lactose, sunflower lecithin (emulsifier), natural vanilla flavor.Testing of P. acidilactici NRRL B-50517 in five types of high-heattreated oil (corn oil, EVOO, LTOO, peanut oil, and vegetable oil)produced results analogous to those observed in peanut butter. Two ofthe oils, EVOO and corn oil (Table 8), displayed impressive survivalrates even after 30 minutes of continuous exposure to 85° C. (Table 9and Table 10). The apparent durability of the probiotic in oil isparticularly conducive to its use in traditional food preparationtechniques which involve heat.

TABLE 8 Survival of P. acidilactici NRRL B-50517 in commercial oil afterhigh temperature (85° C.) treatment Numbers of viable cells after heattreated the integrated P. acidilactici NRRL B-50517 into different typesof oils Oil Type Room % Survival Up to 85° C. % Survival 5 min at 85° C.% Survival Control 2.75E+08 100.00 2.75E+08 100.00 2.75E+08 100.00 ComOil 6.75E+07 24.52 4.55E+07 16.53 6.00E+06 2.18 EVOO 9.70E+07 35.234.20E+07 15.25 1.10E+07 4.00 LTOO 5.90E+07 21.43 3.60E+07 13.08 5.30E+0719.25 Peanut Oil 1.68E+08 61.02 2.30E+07 8.35 0.00E+00 0.00 VegetableOil 1.06E+08 38.50 4.70E+07 17.07 6.00E+06 2.18 * 900 uL oil was heatedto 85° C. on a hot plate before 0.1 g 1 B/g P. acidilactici NRRL B-50517was added, then left for the specified period of time. Tubes were thenremoved and allowed to cool for a minimum of 10 min before seriallydiluting in 0.1% saline and plating on MRS. Plates were incubatedovernight at 45° C. and enumerated the next day. Percent survival wascalculated as a fraction of a saline + P. acidilactici NRRL B-50517control (0.2 g 1 B/g P. acidilactici NRRL B-50517 added to 20 mL 0.1%saline at room temperature). Conclusion: P. acidilactici NRRL B-50517maintains high viable cell counts (CFU/g) in a variety of commercialoils at room temperature and after high temperature treatment.

TABLE 9 Effects on survival of P. acidilactici NRRL B-50517 in Olive oilafter high temperature (85° C.) treatment at different time Numbers ofviable cells after heat EVOO at different treated the integrated P.acidilactici temperature NRRL B-50517 into olive oil % Survival Room1.18E+08 100.00 Up to 85° C. 7.80E+07 66.10 85° C., 5 min 1.90E+07 16.1085° C., 15 min 3.00E+05 0.25 85° C., 30 min 2.00E+05 0.17 *: 900 uL oilwas heated to 85 C. on a hot plate before 0.1 g P. acidilactici NRRLB-50517 was added, then left for the specified period of time. Tubeswere removed from the heat source and allowed to cool for a minimum of10 min before serially diluted and plated onto MRS. Plates wereincubated overnight at 45° C. and enumerated the next day. Percentsurvival at each length of heat treatment was calculated as a fractionof survival in EVOO at room temperature. Conclusion: P. acidilacticiNRRL B-50517 survives high heat treatment in EVOO, producing high viablecell numbers even after 30 min at 85° C. The probiotic would likely becompatible for a variety of food preparation techniques, including thoseinvolving heating.

TABLE 10 Effects on survival of P. acidilactici NRRL B-50517 in corn oilafter high temperature (85° C.) treatment at different time Numbers ofviable cells after heat treated the integrated P. acidilactici % CornOil Temperature NRRL B-50517 into corn oil Survival Room 6.50E+07 100.00Up to 85° C. 6.30E+07 96.92 85° C., 5 min 4.00E+06 6.15 85° C., 15 min5.30E+05 0.82 85° C., 30 min 3.00E+04 0.05 *: 900 uL oil was heated to85 C. on a hot plate before 0.1 g P. acidilactici NRRL B-50517 wasadded, then left for the specified period of time. Tubes were removedfrom the heat source and allowed to cool for a minimum of 10 min beforeserially diluted and plated onto MRS. Plates were incubated overnight at37° C. and enumerated the next day. Percent survival at each length ofheat treatment was calculated as a fraction of survival in corn oil atroom temperature. Conclusions: P. acidilactici NRRL B-50517 surviveshigh heat treatment in corn oil, producing high viable cell numbers evenafter 30 min at 85° C. The probiotic would likely be compatible with avariety of food preparation techniques, including those involvingheating.P. acidilactici NRRL B-50517 can survive after 85° C. heat treateddifferent food products dispenses into the different containers with P.acidilactici NRRL B-50517 simulated sterilization procedures used in thefood industry (Table 11 and Table 12), and retain viability in productswith diverse physiochemical properties for weeks or months. Thisprovides a novel approach to introduce viable probiotics into foods.

TABLE 11 Survival of P. acidilactici NRRL B-50517 in pudding after hightemperature (85° C.) treatment Numbers of viable cells after heattreated the integrated P. acidilactici NRRL B-50517 into different typesof pudding Days after 85° C. Treatment Vanilla pudding Chocolate pudding1 3.55E+07 4.70E+07 2 6.90E+07 2.65E+07 3 6.05E+07 5.10E+07 6 3.60E+071.93E+07 14 3.55E+07 7.75E+06 29 6.10E+07 4.00E+06 *: A 100 mL cup ofShiny Spoon Pudding was emptied into two 50 mL tubes and heated to 85°C. for 20 min. The original container was cleaned with soap and water,dried, and filled with 2 g 10 B/g P. acidilactici NRRL B-50517. Theheated pudding was then poured back into the original container, cooledfor 20 min, then stored in the refrigerator overnight. The next day thepudding was mixed and assayed for viability by first diluting 2 gpudding in 5 mL saline, then serially diluting for plating onto MRS.Plates were incubated overnight and enumerated the following day.Conclusion: After incorporation to either vanilla or chocolate puddingheated to 85° C. in conditions similar to pasteurization, P.acidilactici NRRL B-50517 maintains highly stable cell counts forapproximately one month when stored at refrigerator temperature.

TABLE 12 Survival of P. acidilactici NRRL B-50517 in commercial foodproducts after high temperature (85° C.) treatment P. acidilactici NRRLB-50517 Survival After 85° C. Treatment Food Product CFU/g % SurvivalControl 2.75E+08 100 Ketchup 9.83E+07 35.68 Fruit Cup 8.19E+07 29.75EVOO 7.55E+07 27.42 Great Value Oil 2.43E+07 8.81 2.5% Lactose 2.33E+078.46 Strawberries in Syrup 1.40E+07 5.09 Orange Juice 1.04E+07 3.79 *:Samples were prepared as follows: 1. 10 mL ketchup was heated at 85° C.for 20 min, then mixed with 0.4 g of 1 B/g P. acidilactici NRRL B-50517and 5 mL sterile water and allowed to cool for 20 min before testing forviability. 2. 100 g fruit cup mixture was heated at 85° C. for 45 min,then poured back into the original container over 1 g 1 B/g P.acidilactici NRRL B-50517 and tested for viability. 3. Tubes of 5 mLEVOO and Great Value oil were heated at 85 C. for 20 min, then added to0.2 g 1 B/g P. acidilactici NRRL B-50517 and tested for viability. 4. Atube containing 5 mL 2.5% lactose was heated at 85° C. for 20 min, thenadded to a 15 mL tube containing 0.2 g 1 B/g P. acidilactici NRRLB-50517. The tube was allowed to cool for 20 minutes before testing forviability. 5. 283 g strawberry in syrup mixture was poured into abeaker, heated at 85° C. for 30 min, then poured back into originalcontainer on top of 2 g 1 B/g P. acidilactici NRRL B-50517. Aftercooling for 20 min, the mixture was tested for viability. 6. 5 mL orangejuice was heated to 85° C. for 20 min, then added to 0.1 g 1 B/g P.acidilactici NRRL B-50517, cooled for 20 min and tested for viability.**All viability testing was conducted by serially diluting P.acidilactici NRRL B-50517 + heat-treated food mixture in 0.1% saline,plating onto MRS, and enumerating plates after overnight incubation.Percent survival was calculated as a fraction of a saline + P.acidilactici NRRL B-50517 control (0.2 g 1 B/g P. acidilactici NRRLB-50517 added to 20 mL 0.1% saline at room temperature). Conclusion: P.acidilactici NRRL B-50517 maintains viability in a variety of liquid andsolid matrices after high heat treatment, indicating high compatibilityfor incorporation to many different foods after pasteurization or othersimilar high-heat sterilization procedures.

While there have been shown and described what are presently believed tobe the preferred embodiments of the present invention, those skilled inthe art will realize that other and further embodiments can be madewithout departing from the spirit and scope of the invention describedin this application, and this application includes all suchmodifications that are within the intended scope of the claims set forthherein. All patents and publications mentioned and/or cited herein areincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated as having beenincorporated by reference in its entirety.

What is claimed is:
 1. A method of treating a disease or conditioncharacterized by inflammation in a subject in need thereof, comprisingadministering to the subject an effective amount of Pediococcusacidilactici strain NRRL B-50517 as a probiotic, wherein the method i.stimulates innate immune responses; ii. enhances innate immunity; iii.accelerates weight loss with no changes to diet or exercise patterns;iv. treats pancreatitis; or v. improves a side effect of cancertreatment.
 2. The method of claim 1, wherein the subject is a human. 3.The method of claim 1, wherein the subject is administered greater than1.0×10⁹ cfu of the probiotic.
 4. The method of claim 1, wherein thesubject is administered greater than 4.0×10⁹ cfu of the probiotic. 5.The method of claim 1, wherein the subject is administered one or moreadditional therapeutic agents.
 6. The method of claim 1, wherein thesubject is not administered another therapeutic agent.
 7. The method ofclaim 1, wherein the Pediococcus acidilactici probiotic increases thenumber of anti-inflammatory M2 macrophage cells in the subject.
 8. Themethod of claim 1, wherein the subject exhibits increased IL-10production.
 9. The method of claim 1, wherein the subject exhibitsdecreased levels of IL-6 and/or IL-23.
 10. The method of claim 1,wherein the subject is administered one or more chemotherapeutic agentsand/or radiotherapy in combination with the Pediococcus acidilacticiprobiotic.
 11. A method of treating a disease or condition characterizedby inflammation in a subject in need thereof, comprising administeringto the subject an effective amount of a Pediococcus acidilacticiprobiotic, wherein the method: stimulates innate immune responses; ii.enhances innate immunity; iii. accelerates weight loss with no changesto diet or exercise patterns; iv. treats pancreatitis; or v. improves aside effect of cancer treatment.
 12. The method of claim 11, wherein thesubject is a human.
 13. The method of claim 11, wherein the subject isadministered greater than 1.0×10⁹ cfu of the probiotic.
 14. The methodof claim 11, wherein the subject is administered one or morechemotherapeutic agents and/or radiotherapy in combination with thePediococcus acidilactici probiotic.